Toxoplasma gondii is an important opportunistic infection of AIDS patients. Improved strategies and approaches are urgently needed to more effectively prevent and treat recurrent opportunistic infections in AIDS. Toxoplasma gondii is also being increasingly recognized as a model Apicomplexan parasite and this parasite may be harnessed to serve as a valuable model to investigate the biology of other related protozoan parasites that cause significant human diseases such as malaria and cryptosporidiosis (a significant AIDS OI). We propose to develop improved genetic models to more effectively dissect the fundamental biology of T. gondii and to further develop this model Apicomplexan parasite. We have already developed Type I strains of T. gondii that exhibit a markedly enhanced efficiency of homologous recombination due to an engineered deficiency in a major nonhomologous end-joining DNA repair pathway. These new strains now enable the efficient and reliable construction of directed gene knockouts and gene replacements to study gene function in the Type I genetic background. In specific aim 1 of this proposal we will develop Type II and Type III strains of T. gondii that exhibit a high efficiency of homologous recombination by disrupting the KU80 locus in each strain. In specific aim 2 we will develop a small kit of newly engineered and strain-specific genetic tools that will enhance the ability to conditionally control and genetically manipulate Type I, II and III KU80 knockout strains that now exhibit a high frequency of homologous recombination. These new strains and genetic tools will provide an essential framework for the genetic dissection of strain-specific virulence factors and will enhance biological discovery in the post-genome era by enabling efficient functional genomic studies in all three lineages of Toxoplasma gondii. PUBLIC HEALTH RELEVANCE: This project will develop new genetic models and novel strains of Toxoplasma gondii that can be more easily genetically manipulated. The ability to more easily genetically engineer the model intracellular pathogen Toxoplasma gondii has great potential to more rapidly decipher underlying biology and should lead to new treatments and vaccines to fight significant diseases of humans caused by parasitic protozoa.